Actin filament lengths are precisely regulated and very stable in the red blood cell (RBC) membrane skeleton, while in platelets, actin filament lengths are dynamically regulated during receptor- mediated actin assembly as platelets extend lamellipodia during thrombogenesis. The broad, long term objective of this research is to elucidate how actin filament length and turnover is controlled in RBCs and platelets. An additional objective is to determine the role of actin filament length regulation in RBC membrane skeleton biogenesis and stability, and how aberrant filament length regulation may result in abnormal RBCs and hemolytic anemias. This proposal focuses on molecular mechanisms and in vivo functions of tropomodulins (Tmods), tropomyosin (TM)-regulated actin filament pointed end-capping proteins in RBCs (Tmod1) and platelets (Tmod3). The specific aims are: (1) To investigate the molecular basis for Tmod1 or Tmod3 binding to RBC or platelet TMs, and for TM-regulated actin capping. Biochemical and biophysical assays will be used to identify TM isoform-specific binding and TM-actin capping domains and a novel Tmod3 monomer-binding site. (2) To establish the in vivo function of Tmod1 in regulation of RBC actin filament length, membrane skeleton assembly and stability using mouse models. Tmod1 and Tmod3 conditional knockout mice will be generated followed by breeding with a [unreadable]LCR-[unreadable]pr- Cre mouse to produce single or double knockouts for Tmod1 and/or Tmod3 in RBCs and platelets. RBC phenotypes will be examined by hematology, ektacytometry, membrane skeleton assembly, and electron microscopy of actin filament lengths in membrane skeletons. (3) To investigate an in vivo function for Tmod3 in receptor-mediated actin assembly during activation and spreading of platelets. Platelet function in Tmod3-deficient mouse platelets will be evaluated by hematological analyses, bleeding times, and thrombus formation under flow ex vivo. A role for Tmod3 in actin assembly, shape change and spreading will be evaluated by biochemical assays, immunofluorescence and electron microscopy, and by time-lapse interference reflection and fluorescence microscopy of lamellipodia and filopodia protrusion in living platelets. Molecular requirements for Tmod3 function will be tested using a permeabilized platelet model for receptor-coupled platelet activation, by introduction of Tmod3 or Tmod1 domains and mutants. [unreadable] [unreadable]